Cocaine abuse has long been an epidemic in American society, where in a 1998 National Household Survey on Drug Abuse (NHSDA) it was estimated that 1.8 million Americans age 12 and older were chronic cocaine users. While this number is significantly down from the 1985 NHSDA estimate of 5.7 million users, this has not been the result of an effective medication for the treatment of cocaine abuse, because there is not one. Ongoing research strives to rationally design a drug to treat cocaine abuse centered around its target, the dopamine transporter, but this goal cannot be achieved unless it is fully understood how this protein functions. Without adequate structural information, it is not possible to understand the mechanism of this protein in the absence and presence of drugs of abuse. This proposal seeks to provide the foundation necessary to identify the binding site of this transporter, and hence provide further knowledge of its functional properties, by characterizing a sulfhydryl specific substrate analog, dopamine ortho-quinone (DAQ), for use with wild-type and cysteine mutant constructs of this transporter. The preliminary results for the two aims described in this proposal suggest that it will be possible to identify the amino acids that constitute the transport path of the dopamine transporter, which is a vital step in understanding the mechanism of this transporter.